Integrated analysis of microRNA and mRNA expression profiles in the rat spinal cord under inflammatory pain conditions

Recent studies using microarray‐based approaches have demonstrated that microRNAs (miRNAs) are involved in pain processing pathways. However, a significant proportion of computational predictions of miRNA targets are false‐positive interactions. To increase the chance of identifying biologically relevant targets, we performed an integrated analysis of both miRNA and mRNA expression profiles in the rat spinal cord during complete Freund's adjuvant (CFA)‐induced inflammatory pain. We generated miRNA and mRNA arrays from the same corresponding samples on days 5 and 14 after CFA injection. Five miRNAs and 1096 mRNAs in the CFA 5d group and 16 miRNAs and 647 mRNAs in the CFA 14d group were differentially expressed based on a filter of at least a 1.5‐fold change in either direction. An integrated analysis revealed 54 mRNA targets with an inverse correlation to the expression patterns of three miRNAs in the CFA 5d group. Seventy‐five targets were inversely correlated to six miRNAs in the CFA 14d group. The miRNA–mRNA interaction networks revealed significant changes in miR‐124, miR‐149, miR‐3584 and their target genes, IL‐6R, ADAM19, LAMC1 and CERS2, in the CFA 5d group. In the CFA 14d group, significant changes were noted in miR‐124, miR‐29, miR‐34, miR‐30, miR‐338 and their target genes, TIMP2, CREB5 and EFNB1. We also investigated an interaction pair, miR‐124‐3p and IL‐6R, and the results showed that miR‐124‐3p could attenuate inflammatory pain and decrease IL‐6R expression in the spinal cord. These specific miRNAs and their target genes provide possible avenues for the diagnosis and treatment of inflammatory pain.     

MiRNAs: potential diagnostic and therapeutic targets for cerebral ischaemia

MiRNAs are short single-stranded non-coding RNAs that cause degradation or repression of target mRNAs by base pairing with their 3′-untranslated regions. Recent studies have shown that miRNAs play an important role in the occurrence and development of cerebral ischaemia, as well as exerting regulatory effects. Additionally, circulating miRNAs in peripheral blood, which are dysregulated following cerebral ischaemia, have recently been identified as useful biomarkers in diagnosis and prognosis of cerebral ischaemia. Single-nucleotide polymorphisms (SNPs) located in miRNA genes or target sites are likely to cause complex functional consequences by affecting miRNA biogenesis or target selection. Research on miRNA-SNPs is rapidly growing, and recent studies have identified a significant relationship between miRNAs and ischemic disease. We also address the latest advances in miRNA-based therapeutic approaches for ischemic disease. In conclusion, our review summarizes current research regarding miRNAs and cerebral ischaemia, focusing on the regulatory role of miRNAs in cerebral ischaemia, as well as the potential of miRNAs as biomarkers and therapeutic targets in cerebral ischaemia.     

Molecular network of microRNA targets in Alzheimer's disease brains

MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate translational repression of target mRNAs. The vast majority of presently identified miRNAs are expressed in the brain where they fine-tune the expression of a wide range of target molecules essential for neuronal and glial development, differentiation, proliferation, apoptosis and metabolism. Aberrant expression and dysfunction of brain-enriched miRNAs induce development of neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Because a single miRNA concurrently downregulates hundreds of target mRNAs, the set of miRNA target genes coregulated by an individual miRNA generally constitutes the biologically integrated network of functionally associated molecules. Recent advances in systems biology enable us to characterize the global molecular network of experimentally validated targets for individual miRNAs by using pathway analysis tools of bioinformatics endowed with comprehensive knowledgebase. This review is conducted to summarize accumulating studies focused on aberrant miRNA expression in AD brains, and to propose the systems biological view that abnormal regulation of cell cycle progression as a result of deregulation of miRNA target networks plays a central role in the pathogenesis of AD.     

Target Gene Repression Mediated by miRNAs miR-181c and miR-9 Both of Which Are Down-regulated by Amyloid-β

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that are essential for normal brain development and function. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer’s disease (AD) that is characterized by amyloid-β (Aβ) and tau deposition in brain. How deregulated miRNAs contribute to AD is not understood, as their dysfunction could be both a cause and a consequence of disease. To address this question we had previously profiled miRNAs in models of AD. This identified miR-9 and -181c as being down-regulated by Aβ in hippocampal cultures. Interestingly, there was a remarkable overlap with those miRNAs that are deregulated in Aβ-depositing APP23 transgenic mice and in human AD tissue. While the Aβ precursor protein APP itself is a target of miRNA regulation, the challenge resides in identifying further targets. Here, we expand the repertoire of miRNA target genes by identifying the 3′ untranslated regions (3′ UTRs) of TGFBI, TRIM2, SIRT1 and BTBD3 as being repressed by miR-9 and -181c, either alone or in combination. Taken together, our study identifies putative target genes of miRNAs miR-9 and 181c, which may function in brain homeostasis and disease pathogenesis.     

Association of MicroRNAs with the Clinicopathologic Characteristics of Ependymoma

The current management of ependymoma is wrought with limitations. Molecular classification is a promising development. MicroRNA (miRNA) deregulation is associated with human cancer and may be a means of molecular classification. The aim of our study is to investigate the association of miRNA expression with the clinicopathologic characteristics of ependymoma. Twenty-two samples were clinically annotated. Histologic features were reassessed and the expression of Ki-67, cyclin D1, and nestin was examined. The expression of 84 stem cell-related miRNAs was profiled. The ΔΔC_T method and a Student’s t test were used to compute fold changes and P values, respectively. Our analysis revealed 24 statistically significant associations. We identified seven site-specific miRNAs. The pattern of expression was variable in each anatomic site. In addition, we identified six candidate recurrence biomarkers, all of which were overexpressed in recurrent cases. All three grade-related miRNAs were underexpressed in anaplastic samples. Two miRNAs each were underexpressed in samples immunoreactive to Ki-67 and cyclin D1. No miRNAs were differentially expressed between nestin-negative and nestin-positive samples. In conclusion, molecular alterations in ependymoma involve miRNAs. In our report, we review the level of evidence for the biomarker candidacy of identified miRNAs. Confirmatory studies are necessary to establish robust biomarkers for the clinical management of ependymoma. Proteins regulated by differentially expressed miRNAs are additional candidate biomarkers and may offer targets for novel therapeutic interventions.     

Bioinformatics Analysis of Microarray Profiling Identifies That the miR-203-3p Target Ppp2ca Aggravates Seizure Activity in Mice

This study aimed to identify key genes (microRNA and messenger RNA (mRNA)) and associated signaling-regulated pathways in a drug-induced epilepsy model in mice by microarray profiling. The related microarray dataset of seizures was obtained from the NCBI Gene Expression Omnibus database (GEO), and differentially expressed genes (DEGs) between two control samples or multi-treated samples and samples were analyzed using the statistical software R. To identify the expected function of DEGs, Gene Set Enrichment Analysis (GSEA) was utilized to conduct Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The interaction relationship between microRNAs (miRNAs) and mRNAs in normal and epilepsy mouse models was identified using Cytoscape software. TargetScan7.1 was applied to determine the binding sites of DEGs. The dual-luciferase assay was used to verify the target relationship between miRNA and mRNA. Four miRNAs were identified as differentially expressed genes in both 24-h and 28-day status epilepticus (SE)-treated samples. Ppp2ca expression in the mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the pilocarpine-induced SE mouse model. The expression of Ppp2ca was also downregulated in the kinase-induced SE model group compared with that in the untreated group and MAP kinase (MEK) inhibitor-treated group of mice. KEGG pathway analysis indicated that the MAPK signaling pathway was upregulated in the kinase-induced SE model group compared with that in both the untreated group and the MEK inhibitor-treated group of mice. miR-203 had a targeted relationship with Ppp2ca in both humans and mice. The miR-203-3p target Ppp2ca aggravates the seizures of the SE model in mice.     

Aberrant microRNA expression in the brains of neurodegenerative diseases: miR‐29a decreased in Alzheimer disease brains targets neurone navigator 3

M. Shioya, S. Obayashi, H. Tabunoki, K. Arima, Y. Saito, T. Ishida and J. Satoh (2010) Neuropathology and Applied Neurobiology 36, 320–330
Aberrant microRNA expression in the brains of neurodegenerative diseases: miR‐29a decreased in Alzheimer disease brains targets neurone navigator 3 Aims: MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translational repression of target mRNAs. Accumulating evidence indicates that various miRNAs, expressed in a spatially and temporally controlled that manner in the brain plays a key role in neuronal development. However, at present, the pathological implication of aberrant miRNA expression in neurodegenerative events remains largely unknown. To identify miRNAs closely associated with neurodegeneration, we performed miRNA expression profiling of brain tissues of various neurodegenerative diseases. Methods: We initially studied the frontal cortex derived from three amyotrophic lateral sclerosis patients by using a microarray of 723 human miRNAs. This was followed by enlargement of study population with quantitative RT‐PCR analysis (n = 21). Results: By microarray analysis, we identified up‐regulation of miR‐29a, miR‐29b and miR‐338‐3p in amyotrophic lateral sclerosis brains, but due to a great interindividual variation, we could not validate these results by quantitative RT‐PCR. However, we found significant down‐regulation of miR‐29a in Alzheimer disease (AD) brains. The database search on TargetScan, PicTar and miRBase Target identified neurone navigator 3 (NAV3), a regulator of axon guidance, as a principal target of miR‐29a, and actually NAV3 mRNA levels were elevated in AD brains. MiR‐29a‐mediated down‐regulation of NAV3 was verified by the luciferase reporter assay. By immunohistochemistry, NAV3 expression was most evidently enhanced in degenerating pyramidal neurones in the cerebral cortex of AD. Conclusions: These observations suggest the hypothesis that underexpression of miR‐29a affects neurodegenerative processes by enhancing neuronal NAV3 expression in AD brains.     

Harnessing Cerebrospinal Fluid Biomarkers in Clinical Trials for Treating Alzheimer's and Parkinson's Diseases: Potential and Challenges

No disease-modifying therapies (DMT) for neurodegenerative diseases (NDs) have been established, particularly for Alzheimer''s disease (AD) and Parkinson''s disease (PD). It is unclear why candidate drugs that successfully demonstrate therapeutic effects in animal models fail to show disease-modifying effects in clinical trials. To overcome this hurdle, patients with homogeneous pathologies should be detected as early as possible. The early detection of AD patients using sufficiently tested biomarkers could demonstrate the potential usefulness of combining biomarkers with clinical measures as a diagnostic tool. Cerebrospinal fluid (CSF) biomarkers for NDs are being incorporated in clinical trials designed with the aim of detecting patients earlier, evaluating target engagement, collecting homogeneous patients, facilitating prevention trials, and testing the potential of surrogate markers relative to clinical measures. In this review we summarize the latest information on CSF biomarkers in NDs, particularly AD and PD, and their use in clinical trials. The large number of issues related to CSF biomarker measurements and applications has resulted in relatively few clinical trials on CSF biomarkers being conducted. However, the available CSF biomarker data obtained in clinical trials support the advantages of incorporating CSF biomarkers in clinical trials, even though the data have mostly been obtained in AD trials. We describe the current issues with and ongoing efforts for the use of CSF biomarkers in clinical trials and the plans to harness CSF biomarkers for the development of DMT and clinical routines. This effort requires nationwide, global, and multidisciplinary efforts in academia, industry, and regulatory agencies to facilitate a new era.     

Hypericin as a potential drug for treating Alzheimer's disease and type 2 diabetes with a view to drug repositioning

Aims

Alzheimer's disease (AD) and type 2 diabetes (T2D) are two of the most common diseases in elderly population and they have a high rate of comorbidity. Study has revealed that T2D is a major risk factor of AD, and thus exploring therapeutic approaches that can target both diseases has drawn much interest in recent years. In this study, we tried to explore drugs that could be potentially used to prevent or treat both AD and T2D via a drug repositioning approach.

Methods

We first searched the known drugs that may be effective to T2D treatment based on the network distance between the T2D-associated genes and drugs deposited in the DrugBank database. Then, via molecular docking, we further screened these drugs by examining their interaction with islet amyloid polypeptide (IAPP) and Aβ42 peptide, the key components involved in the pathogenesis of T2D or AD. Finally, the binding between the selected drug candidates and the target proteins was verified by molecular dynamics (MD) simulation; and the potential function of the drug candidates and the corresponding targets were analyzed.

Results

From multiple resources, 734 T2D-associated genes were collected, and a list of 1109 drug candidates for T2D was obtained. We found that hypericin had the lowest binding energy and the most stable interaction with either IAPP or Aβ42 peptide. In addition, we also found that the target genes regulated by hypericin were differentially expressed in the tissues related to the two diseases.

Conclusion

Our results show that hypericin may be able to bind with IAPP and Aβ42 stably and prevent their accumulation, and thus could be a promising drug candidate for treating the comorbidity of AD and T2D.     

Advances and applications of fluids biomarkers in diagnosis and therapeutic targets of Alzheimer's disease

Aims

Alzheimer's disease (AD) is a neurodegenerative disease with challenging early diagnosis and effective treatments due to its complex pathogenesis. AD patients are often diagnosed after the appearance of the typical symptoms, thereby delaying the best opportunity for effective measures. Biomarkers could be the key to resolving the challenge. This review aims to provide an overview of application and potential value of AD biomarkers in fluids, including cerebrospinal fluid, blood, and saliva, in diagnosis and treatment.

Methods

A comprehensive search of the relevant literature was conducted to summarize potential biomarkers for AD in fluids. The paper further explored the biomarkers' utility in disease diagnosis and drug target development.

Results

Research on biomarkers mainly focused on amyloid-β (Aβ) plaques, Tau protein abnormal phosphorylation, axon damage, synaptic dysfunction, inflammation, and related hypotheses associated with AD mechanisms. Aβ₄₂, total Tau (t-Tau), and phosphorylated Tau (p-Tau), have been endorsed for their diagnostic and predictive capability. However, other biomarkers remain controversial. Drugs targeting Aβ have shown some efficacy and those that target BACE1 and Tau are still undergoing development.

Conclusion

Fluid biomarkers hold considerable potential in the diagnosis and drug development of AD. However, improvements in sensitivity and specificity, and approaches for managing sample impurities, need to be addressed for better diagnosis.     

Chemokine receptor 7 mediates miRNA-182 to regulate cerebral ischemia/reperfusion injury in rats

Aims

Chemokine receptor 7 (CXCR7) exerts protective effects on the brain. MicroRNAs (miRNAs) are involved in cerebral ischemia/reperfusion (I/R) injury, but their involvement in CXCR7-mediated brain protection is unknown. In this study, we investigated the role of miRNAs in CXCR7-mediated brain protection.

Methods

CXCR7 levels in peripheral blood samples from patients with acute ischemic stroke (AIS) and ischemic penumbra area brain tissues from middle cerebral artery occlusion (MCAO) rats after recanalization were measured. An miRNA microarray analysis was performed to examine the expression of miRNAs caused by CXCR7 knockdown in ischemic penumbra area brain tissue in middle cerebral artery occlusion–reperfusion rats and to predict corresponding downstream target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed the most enriched pathways. A dual-luciferase reporter assay confirmed the direct regulation of miR-182 on the target gene TCF7L2. The correlation between TCF7L2 and CXCR7/miR-182 was verified using rescue assays.

Results

CXCR7 expression was upregulated in MCAO rats and mechanical thrombectomy patients with AIS compared to that in controls. The motor and sensory functions of MCAO rats with CXCR7 knockdown further decreased, and the infarct volume and cerebral edema increased. miRNA microarray data showed that seven miRNAs were differentially expressed after shRNA-CXCR7 treatment. The dual-luciferase reporter assay confirmed that miR-182 directly targeted the TCF7L2 gene. Rescue assays confirmed that TCF7L2 is downstream of CXCR7/miR-182. KEGG pathway analysis showed that the Hippo pathway may be a key pathway in CXCR7 upregulation and plays a role in protecting the brain after interventional surgery. Animal experiments have shown that CXCR7-mediated cerebral I/R injury promotes the phosphorylation of key molecules YAP and TAZ in the Hippo pathway.

Conclusion

CXCR7 protects against cerebral I/R injury, possibly via the miR-182/TCF7L2/Hippo pathway. These results indicate that CXCR7 affects cerebral ischemia–reperfusion injury through miRNA regulation and downstream pathways.     

Multiplex Analysis of Cytokines in the Serum and Cerebrospinal Fluid of Patients With Alzheimer's Disease by Color-Coded Bead Technology

Background and purpose

The availability and promise of effective treatments for neurodegenerative disorders are increasing the importance of early diagnosis. Having molecular and biochemical markers of Alzheimer''s disease (AD) would complement clinical approaches, and further the goals of early and accurate diagnosis. Combining multiple biomarkers in evaluations significantly increases the sensitivity and specificity of the biochemical tests.

Methods

In this study, we used color-coded bead-based Luminex technology to test the potential of using chemokines and cytokines as biochemical markers of AD. We measured the levels of 22 chemokines and cytokines in the serum and cerebrospinal fluid (CSF) of 32 de novo patients (13 controls, 11 AD, and 8 Parkinson''s disease [PD]).

Results

MCP-1 was the only cytokine detectable in CSF, and its levels did not differ between control and disease groups. However, the serum concentration of eotaxin was significantly higher in AD patients than in the control group.

Conclusions

The analysis of multiple inflammatory mediators revealed marginal differences in their CSF and serum concentrations for the differential diagnosis of AD and PD. These results provide evidence that immunological responses are not major contributors to the pathogenesis of AD and PD.